In the process of culturing primary fibroblast, my control group showed high level of a-SMA in subsequent Western blot, however, I need to confirm the activation effect of TGF-b, and the anti-activation effect of a drug.
To avoid self-activation of primary lung fibroblasts during culture, several strategies can be employed:
1. Optimize Culture Conditions: Ensure that the culture medium is free from factors that could induce fibroblast activation, such as excessive serum. Try using a low-serum medium or serum-free conditions if possible.
2. Maintain Confluency: Over-confluence can induce fibroblast activation. It’s essential to maintain fibroblasts at a lower confluency (around 70–80%) to prevent spontaneous activation.
3. Use Specific Inhibitors: Adding specific inhibitors of the TGF-β pathway (e.g., SB431542) to the culture can help prevent the spontaneous activation of fibroblasts into myofibroblasts, as TGF-β is a key player in fibroblast activation.
4. Subculture Promptly: Avoid prolonged culture periods, as fibroblasts can undergo activation over time. Regular subculturing can help maintain their quiescence.
5. Proper Controls: Ensure that your control group is cultured under identical conditions without TGF-β stimulation. You can include non-treated cells in every experiment as a negative control to detect spontaneous activation.
Confirming the activation effect of TGF-β and the anti-activation effect of a drug would involve using TGF-β as a positive control to induce activation (e.g., increased α-SMA expression) and testing your drug under the same conditions.
These methods can help reduce the self-activation and ensure more reliable results for your experiments.
I agreed what the strategies Gideon mentioned. Based on my experience in culture skin fibroblasts, to avoid self activation, change medium daily is critical.
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